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Biomaterials 32 (2011) 2748e2756

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Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

Recapitulation of the embryonic cardiovascular progenitor cell niche

Katja Schenke-Layland a,b,c,*, Ali Nsair c, Ben Van Handel d,e, Ekaterini Angelis c, Jessica M. Gluck c,Miriam Votteler a,b, Joshua I. Goldhaber f, Hanna K. Mikkola d,e, Michael Kahn g,h, i,William R. MacLellan c,e

aDepartment of Cell and Tissue Engineering, Fraunhofer IGB, 70569 Stuttgart, GermanybCenter for Regenerative Biology and Medicine, Eberhard Karls University Tübingen, 72076 Tübingen, GermanycDepartment of Medicine/Cardiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USAdDepartment of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA 90095, USAe Edythe and Eli Broad Center for Regenerative Medicine and Stem Cell Research at UCLA, University of California, Los Angeles, CA 90095, USAfCedars-Sinai Medical Center, Department of Medicine, Los Angeles, CA 90048, USAg Edythe and Eli Broad Center for Regenerative Medicine and Stem Cell Research at USC, University of Southern California, Los Angeles, CA 90033, USAhDepartment of Biochemistry and Molecular Biology, Center for Molecular Pathways and Drug Discovery, University of Southern California, Los Angeles, CA 90033, USAiDepartment of Molecular Pharmacology and Toxicology, Center for Molecular Pathways and Drug Discovery, University of Southern California, Los Angeles, CA 90033, USA

a r t i c l e i n f o

Article history:Received 9 December 2010Accepted 28 December 2010Available online 22 January 2011

Keywords:Extracellular matrixCardiac tissue engineeringStem cellScaffoldHeart

* Corresponding author. Fraunhofer-Institute forBiotechnology (IGB), Nobelstr. 12, 70569 Stuttgart, Gerfax: þ49 711970 4158.

E-mail address: katja.schenke-layland@igb.fraunh

0142-9612/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.biomaterials.2010.12.046

a b s t r a c t

Stemor progenitor cell populations are often established in unique nichemicroenvironments that regulatecell fate decisions. Although niches have been shown to be critical for the normal development of severaltissues, their role in the cardiovascular system is poorly understood. In this study, we characterized thecardiovascular progenitor cell (CPC) niche in developing human and mouse hearts, identifying signalingpathways and extracellular matrix (ECM) proteins that are crucial for CPCmaintenance and expansion.Wedemonstrate that collagen IV (ColIV) and b-catenin-dependent signaling are essential for maintaining andexpanding undifferentiated CPCs. Since niches are three-dimensional (3D) structures, we investigated theimpact of a 3D microenvironment that mimics the in vivo niche ECM. Employing electrospinning tech-nologies, 3D in vitro niche substrates were bioengineered to serve as culture inserts. The three-dimen-sionality of these structures increased mouse embryonic stem cell differentiation into CPCs whencompared to 2D control cultures, which was further enhanced by incorporation of ColIV into thesubstrates. Inhibiting p300-dependent b-catenin signals with the small molecule IQ1 facilitated furtherexpansion of CPCs. Our study represents an innovative approach to bioengineer cardiac niches that canserve as unique 3D in vitro systems to facilitate CPC expansion and study CPC biology.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

The mammalian heart is the first organ to develop and functionin the fetus. It contains three major cell lineages, cardiac myocytes(CMs), smooth muscle cells (SMCs) and endothelial cells (ECs),which are believed to derive from a common cardiovascularprogenitor cell (CPC) [1e4]. This progenitor represents one of theearliest stages in mesodermal specification to the cardiovascularlineage. It can be identified in mouse embryos or embryonic stem(ES) cell derivatives by expression of the kinase insert domainprotein receptor-expressing (KDR, also known as Flk1). Subsequent

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studies have reported additional markers, defining multipotentmouse CPCs including Islet1 (Isl1þ) and NK2 transcription factor-related, locus 5 (Nkx2.5þ) [1e3]. We isolated Flk1þ progenitor cellsfrom differentiating mouse ES or induced-pluripotent stem (iPS)cells that possess the ability to differentiate into all three cell typesof the cardiovascular lineage as well as hematopoietic cells [5].A comparable CPC is also present during human cardiogenesis [6];however, this Isl1þ/Flk1þ progenitor is rapidly lost postnatally andis not detectable in the adult heart.

Endogenous Isl1þ CPCs are found in discrete clusters within thedeveloping right ventricle, the atria and outflow tracts of bothhuman andmouse hearts [3,6], which are reminiscent of a stem cellniche. Niches are anatomically defined, three-dimensional (3D)microenvironments that regulate stem or progenitor cell fatethrough cellecell interactions, cellematrix contacts and localizedsoluble factors [7e9]. Significant progress has been made in char-acterizing such specific microenvironments in selected stem cell

K. Schenke-Layland et al. / Biomaterials 32 (2011) 2748e2756 2749

compartments, including the hematopoietic, epidermal, intestinsaland neural stem cell niches; however, the characteristics of thecardiovascular niche are poorly defined [10]. A major challenge instem cell biology is defining the components of such niches that arecrucial for regulating stem/progenitor cell development andmaintenance, as well as exploiting this knowledge for therapeuticpotential. The interplay between stem or progenitor cells and theirniche creates a dynamic system necessary for maintaininga balance between self-renewal and differentiation of the cells.Wnt/b-catenin signaling is important for the maintenance and self-renewal of stem/progenitor cells of various lineages and has alsobeen implicated in cardiac development [11]. In the cardiovascularsystem, Wnt signaling through b-catenin appears to regulate boththe specification and the proliferation of Isl1þ CPCs [12]. b-catenindirectly binds and regulates the Isl1 promoter in cardiovascularcells [13]. Ablation of b-catenin in Isl1þ cells lead to the disruptionof multiple aspects of cardiogenesis including a defective outflowtract and right ventricular development, resulting in embryoniclethality [13,14]. These data strongly suggest that b-cateninsignaling plays an important role in the specification and mainte-nance of Isl1þ CPCs.

Extracellular matrix (ECM) modulates cell anchorage andactivity of signaling molecules by direct interaction with stem cellsand their progeny [7,8,15]. The ECM of the heart is generallycomposed of collagens, particularly collagen types I (ColI; equalsw85% of the cardiac collagens), III, IV (ColIV) and VI; fibronectin,elastin, glycoproteins and proteoglycans [8,16]. These ECMcomponents are expressed in precise temporal and spatial patterns,and are constantly remodeled during fetal cardiac developmentand in diseased states [17]. While fibrillar collagens like ColI playamajor role in disease-related remodeling processes [18], ColIV andseveral glycoproteins, including laminins, are involved in cardio-vascular development and have been implicated to contribute tothe stem cell niche in the hematopoietic and nervous systems [19].ColIV and laminin have also been identified to regulate cardiac stemcells in the adult heart [10,17]; however, their role in regulating thecardiovascular niche in the developing heart remains unclear.

To determine if CPCs also residewithin a niche in the developingheart, we examined fetal human and murine hearts for the pres-ence of an anatomically distinct niche and attempted to charac-terize the ECM molecules and signaling pathways that mediate itsinteractions with CPCs. We demonstrate that the cardiovascularniche is enriched for the ECM protein ColIV, which plays a key rolein controlling CPC fate. In addition, we show that inhibiting p300-dependent b-catenin signaling is critical for CPC in vitro expansion.To recapitulate the 3D cardiovascular niche microenvironment invitro, we created unique electrospun scaffolds and demonstratethat three-dimensionality itself supports CPC expansion.

2. Materials and methods

2.1. Human and murine tissue procurement and processing

First trimester (6e12 weeks of developmental age) human fetal hearts werediscarded material obtained from elective terminations of pregnancies performedby Family Planning Associates in Los Angeles. Mouse embryos were isolated fromCF1 mice at E15.5 gestational age. All heart tissues were immediately washed afterharvest in sterile phosphate buffered saline (PBS, HyClone, Logan, UT), fixed in 10%buffered formalin for 12 h, then rinsed in tap water and transferred to 70% ethanol.Fixed specimens were embedded in paraffin and cut into 5 mm sections by the UCLATranslational Pathology Core Laboratory (TPCL). Hematoxylin/eosin staining wasperformed by TPCL.

2.2. Immunohistochemistry

Slides were deparaffinized and subjected to antigen retrieval (95 �C for 30 mineach in 10 mM Tris, 1 mM EDTA, 0.05% Tween 20 solution, pH 9.0 followed by 10 mM

citrate solution in PBS, pH 6.0). Endogenous peroxidases were quenched in a 0.9%

solution of H2O2 in methanol for 20 min at room temperature (RT). All sections werethen exposed to blocking buffer (5% horse serum, 0.05% Tween 20 in PBS) for 15min.Primary antibodies were diluted in blocking buffer and applied over night at 4 �Cfollowed by incubation with biotinylated secondary antibodies for 30 min at RT.A complete list of all primary antibodies is provided in Supplemental Table 1.Secondary antibodies (1:500; all from Vector Labs, Burlingame, CA) were detectedusing the ABC system as directed (Vector Labs). For mouse primary antibodies onmouse tissues, the M.O.M. kit (BMK-2202, Vector Labs) was used as directed. Bright-field images were acquired using a Zeiss Axiovert 200 microscope (Carl ZeissMicroImaging, Inc., Thornwood, NY).

2.3. Immunofluorescence staining and confocal microscopy

Unstained sections were deparaffinized and rehydrated. Antigen retrieval wasperformed and all slides were further processed as described before [20]. Aftersecondary antibody incubation, all slides were exposed to DAPI solution (D8417,5 mg/ml in PBS, Sigma Aldrich, St. Louis, MO) for 5 min followed by mounting usingProLong Gold antifade mounting medium (Molecular Probes, Carlsbad, CA). Fluo-rescence images were acquired using a confocal TCS SP2 AOBS laser-scanningmicroscope system (Leica Microsystems Inc., Mannheim, Germany). Images wereprocessed with Adobe Photoshop CS4 (Adobe Systems Inc., San Jose, CA).

2.4. Mouse ES cell cultures

D3 ES cells (CRL-1934, ATCC, Manassas, VA) were maintained in an undiffer-entiated state on mitomycin-C-treated (M4287, Sigma), primary mouse embryonicfibroblasts (MEFs) in leukemia inhibitory factor (LIF) supplemented medium[Knockout Dulbecco’s modified Eagle’s medium supplemented with 15% ES cell-qualified fetal calf serum (ES-FCS), 0.1 mM b-mercaptoethanol, 2 mM glutamine,0.1 mM nonessential amino acids (all from Invitrogen, Carlsbad, CA) and 1000 U/mlrecombinant LIF (Chemicon, Temecula, CA)] at 37 �C, 5% CO2 as described before [5].aMHC-luciferase expressing CGR8 ES cells (alpha-MHC-Luc ES cells; a kind gift ofRichard T. Lee, Harvard Medical School [21]) were maintained MEF-free on 0.1%gelatin (G1890, Sigma) in LIF supplemented medium at 37 �C, 5% CO2. The mediumwas changed on a daily base.

2.5. In vitro differentiation experiments

To remove MEFs, D3 ES cells were collected by trypsinization and plated 2 � on0.1% gelatin-coated culture dishes for 20 min at 37 �C. All nonadherent cells wereused for further experiments. ES cells were then either introduced into a dynamicsuspension culture system for generating spontaneously differentiating embryoidbodies (EBs) or they were cultured on 0.1% gelatin- or extracellular matrix-coatedplates and flasks as described before in detail [5]. Collagen type I- and IV-, laminin-and fibronectin-coated labware was purchased from BD Biocoat (BD Biosciences,Bedford, MA). To induce differentiation into cardiovascular progenitors, all cellswere cultured in LIF-free a-minimum essential medium (Invitrogen), supplementedwith 10% ES-FCS, 0.1 mM b-mercaptoethanol, 2 mM glutamine, and 0.1 mM nones-sential amino acids (a-MEM). For CPC expansion, a-MEMwas further supplementedwith IQ1 (4 mg/ml; [22]).

2.6. Magnetic cell sorting and CPC differentiation

To induce differentiation into Flk1-positive cardiovascular progenitors, undif-ferentiated aMHC-Luc ES cells were trypsinized and cultured in a-MEM for 4 days onColIV-coated flasks as described before [5]. Cells were harvested and the Flk1-positive CPCs were isolated by indirect magnetic cell sorting (MACS) using a purifiedrat anti-mouse Flk1 antibody (550549 [1:200]; BD Pharmingen, San Diego, CA) andmagnetic microbeads (Miltenyi Biotec, Auburn, CA). Flk1-positive CPCs were thenexposed for 4 days to collagen type I- and IV-, laminin- and fibronectin in a-MEM.

2.7. Flow cytometry

Fluorescence-activated cell sorter (FACS) analysis was performed as describedbefore [5]. Cells were stained using phycoerythrin (PE)-conjugated monoclonal ratanti-mouse Flk1 antibody (12-5821 [1:200]; eBioscience Inc., San Diego, CA). A PE-conjugated rat IgG2a k isotype (12-4321 [1:200]; eBioscience) served as control.Staining with 7-aminoactinomycin D (559925; BD Pharmingen, San Diego, CA) wasperformed to exclude dead cells. Cells were analyzed on a BD LSR II cytometer (BDBiosciences). Data analysis was performed using FlowJo 8.6.3 software (Tree StarInc., Ashland, OR).

2.8. Luciferase assays

aMHC promoter activity was assessed with a luciferase assay kit (PromegaCorporation, Madison, WI). Briefly, after the culture mediumwas removed, the cellswere washed once with 1 � PBS and lysed with ice-cold 300 ml of reporter lysisbuffer. 200 ml of the cell lysate was then added to 50 ml of luciferase substratesolution. Bioluminescence generated was measured for 2 s using a Monolight 3010

K. Schenke-Layland et al. / Biomaterials 32 (2011) 2748e27562750

luminometer (BD Pharmingen). The luminescence readings obtained werenormalized to the protein content of the corresponding cell lysate determined bya Bradford assay according to the manufacturer’s protocol (BioRad, Hercules, CA).Results are depicted in relative light units (RLU).

2.9. Gene expression analysis

Total RNA was extracted from cells using the RNeasy Plus Mini Kit as per manu-facturer’s instructions (Qiagen, Valencia, CA). First strand cDNA was generated from2 mg of total RNA by using the Omniscript� Reverse Transcriptase Kit (Qiagen). Semi-quantitative RT-PCR was performed as previously described [5]. The sequences ofeach primer set, including their annealing temperature and cycles, are published [5].Quantitative real-time PCR was performed as previously described [23]. Primer setsspecific for mouse Flk1 (QT00097020) and glyceraldehyde-3-phosphate dehydro-genase (GAPDH;QT01658692)wereobtained fromQiagen (QuantiTect PrimerAssay).

RNA samples from undifferentiated mouse ES cells and mouse ES cell-derivedFlk1� cells and Flk1þ CPCs were analyzed at the UCLA Illumina Microarray Labo-ratory. Briefly, biotinylated cRNAwas prepared using the Illumina RNA AmplificationKit (Ambion Inc., Austin, TX). Samples were hybridized on a Sentrix MouseRef-8Expression BeadChip System (Illumina Inc., San Diego, CA). Scanning was performedaccording to the Illumina BeadStation 500 manual. Microarray raw data wereanalyzed using BeadStudio software version 1.5.1.3. Differential expression analysiswas selected to quantify gene expression intensity values as well as to determinechanges of the gene expression levels between undifferentiated ES cells (referencegroup) and Flk1� cells and Flk1þ CPCs. To filter out nonspecific signal intensities,local background subtraction was performed. Only genes with intensities >0.99were selected for analysis. A differential score of 13 demonstrated that geneexpression from differentiated mouse ES cells had changed significantly whencompared with genes of undifferentiated ES cells.

2.10. Fabrication of electrospun substrates

To fabricate 3D cell culture inserts, a solution containing 10% gelatin (G1890,Sigma) and 10% polycaprolactone (PCL; 19561, Polysciences, Inc., Warrington, PA) in1,1,1,3,3,3-hexafluoro-2-propanol (HFP; 105228, Sigma) was exposed to electro-spinning as described before [24]. After fabrication, the 3D electrospun substrates(300e400 mm thick) were adhered to the bottom of a standard 24-well tissueculture plate with sterile Silastic Medical Adhesive, Type A (3244393, Dow Corning,Midland, MI), followed by disinfection using 70% ethanol for 20 min followed by3 � 5 min rinses in sterile water and PBS. Some of the substrates were then coatedfor 1 h at RT using collagen type IV (354233, BD Biosciences) at a concentration of100 mg/ml (diluted in 0.05 N Hydrochloric acid (H1758, Sigma)). All electrospunsubstrates (coated and uncoated controls) were subsequently analyzed usingscanning electron microscopy (SEM) as described before [24]. Fiber diameters andpore sizes were measured on SEM images. Average fiber diameters were determinedfrom measurements taken perpendicular to the long axis of the fibers withinrepresentative microscopic fields (20 measurements per field). The pores formed atthe interstices of the fibers were measured using ImageJ software (free downloadavailable at http://rsbweb.nih.gov/ij/). For each sample, at least 5 scanning electronmicrographs at 2000 � magnification were used for image analysis and pore sizemeasurements. Immunofluorescence imaging was performed as described above totest if the collagen type IV-coating was carried out successfully.

2.11. Statistical analyses

All data are presented as mean� standard deviation (SD). Statistical significancewas assessed by Student’s t test or ANOVA with Tukey’s multiple comparison testsusing StatView software. P-values less than 0.05 were defined as statisticallysignificant.

3. Results

3.1. Characterization of the endogenous CPC niche

Multipotent endogenous CPCs have been identified in the fetalmouse heart based on the expression of the cardiogenic tran-scription factor Isl1 [2,3]. We have previously described a system topotentiate differentiation of murine ES cells to CPCs based onculturing them on ColIV. These Flk1þ progenitor cells express Isl1 aswell as other known CPC markers including Nkx2.5 (Suppl. Fig. 1A)and possess the ability to differentiate into functional cardiovas-cular cells, including CMs, SMCs and ECs (Suppl. Figs. 2 and 3), aspreviously described [5]. Immunofluorescence imaging of humanfirst trimester (6e12 weeks of developmental age) and murineE15.5 hearts revealed that the endogenous cardiac Isl1þ cells also

show strong expression of Flk1 (Fig. 1A). These Isl1þ/Flk1þ CPCs arelocalized into discrete clusters within the right ventricular freewall, atria and outflow tracks (Fig. 1B and C). Interestingly, immu-nohistochemical staining demonstrated that differentiating CPCs,which migrate away from the progenitor clusters, down-regulateIsl1 while up-regulating mature cardiac markers such as troponin C(Fig. 1C). To determine if the clusters of Isl1þ/Flk1þ cells occupya distinct niche and to characterize its ECM composition, weemployed immunofluorescence staining. Basement membrane(BM) proteins ColIV and laminin were found within and circum-scribing the Isl1þ/Flk1þ CPC clusters, reminiscent of stem/progen-itor cell niches in other tissues (Fig. 1D, E). In contrast, ColI andfibronectin were predominantly found outside the CPC clusterswithin the myocardium (Fig. 1F, G).

3.2. ES cell-derived CPCs and ECM

It has been suggested that ECM proteins are capable of directingES cell differentiation to mesodermal fates, including the cardio-vascular lineages [5,20,25]. We wanted to determine if ColIV andlamininwere associated with Isl1þ/Flk1þ cells in differentiating EBsat time points when commitment to the cardiovascular lineageoccurs. As expected, cells in differentiating EBs down-regulatedpluripotency markers such as Oct4 and Nanog and up-regulatedearly mesodermal (brachyury), endodermal (alpha-fetoprotein)and ectodermal (nestin) markers (Suppl. Fig. 4). We found that ECMproteins ColI and IV, laminin and fibronectin were robustlyexpressed (Suppl. Fig. 5) and co-localized with Isl1þ/Flk1þ cellswithin developing EBs (Fig. 1H and I; Suppl. Fig. 6).

3.3. Impact of ECM on cardiovascular fate

To determine the effects of the ECM proteins on cardiovasculardifferentiation, we cultured wild type mouse ES cells as well asalpha-MHC-Luc ES cells on ECM protein-coated, conventional 2Dculture surfaces (Fig. 2A). We found that ES cells, cultured for 4 dayson the ColIV and laminin, showed significantly increased Flk1 geneexpression when compared to ES cells that had been cultured oneither ColI or fibronectin (Fig. 2B). FACS analyses also demonstratedthat ColIV (2.13 � 0.29%; P < 0.002) significantly increased thepercentage of Flk1þ candidate CPCs when compared to ColI(0.71�0.09%), laminin (0.98� 0.05%) and fibronectin (0.8� 0.01%).To explore the effect of the BM ECM proteins on cardiac differen-tiation, we exposed alpha-MHC-Luc ES cells to ColI, ColIV, lamininand fibronectin. Similar to the results with Flk1, luciferase activitywas 2-fold higher in cells cultured on ColIV and laminin (Fig. 2C). Todetermine if this increase in cardiac differentiationwas due to ColIVand laminin increasing the number of Flk1þ CPCs versus an abilityof ColIV and laminin to enhance cardiac differentiation of Flk1þ

CPCs, we isolated Flk1þ cells from the alpha-MHC-Luc ES cells andthen differentiated them on the various ECM substrates (ColI, ColIV,laminin or fibronectin) (Fig. 2D). Purified, alpha-MHC-Luc ES cell-derived Flk1þ CPCs cultured on ColI, ColIV, or laminin showeda similar propensity for cardiac differentiation as measured byalpha-MHC-dependent luciferase activity (Fig. 2E). Only fibro-nectin, an ECM protein that we found to be localized outside of theputative CPC niche, enhanced differentiation of Flk1þ CPCs intocardiac myocytes (Fig. 2E).

3.4. b-Catenin signaling and CPC fate

The Wnt/b-catenin pathway has been shown to play an impor-tant role in the self-renewal and subsequent differentiation of Isl1þ

CPCsderived frommouseES cells andhearts [12,13]. Consistentwiththese findings, endogenous Isl1þ/Flk1þ CPCs in the cardiovascular

Fig. 1. (A) Immunofluorescence staining of 9 week human hearts reveals that endogenous Isl1þ CPCs (red) also express Flk1 (green). DAPI-stained cell nuclei are shown in white.(BeC) Immunohistochemistry shows that endogenous Isl1þ CPCs are localized in clusters (B, see arrows). Differentiating CPCs down-regulate Isl1 (blue) and up-regulate maturecardiac markers such as troponin C (pink) (C). (DeG) ColIV (D; green) and laminin (E; green) are tightly expressed around the CPC clusters, whereas ColI (F; green) and fibronectin(G; green) are predominantly expressed outside the niche within the myocardium. Cell nuclei are shown in blue (DAPI). (HeI) Flk1þ and Isl1þ CPCs (red) co-localized with ColIV(green) in differentiating mouse embryoid bodies. DAPI-stained nuclei are shown in blue.

K. Schenke-Layland et al. / Biomaterials 32 (2011) 2748e2756 2751

niche in fetal human and mouse hearts express high levels ofb-catenin (Fig. 3A). To determine if genes within theWnt/b-cateninsignaling pathway were preferentially expressed in CPCs, weanalyzed global gene expression in mouse ES cell-derived Flk1þ

progenitors compared to Flk1� cells using microarrays. Wntsignaling molecules, wingless-type MMTV integration site 9A(Wnt9a) and wingless-related MMTV integration site 1 (Wnt1) aswell as Wnt membrane receptor frizzled homolog 4 (Fzd4) werepreferentially expressed in Flk1þ cells (Table 1). b-catenin (Ctnnb1)as well SRY-box containing gene 7 (Sox7) and myelocytomatosisoncogene (c-myc), both b-catenin targets were up-regulated(Table 1). Consistent with activation of theWnt/b-catenin pathway,negative regulators such as Wnt inhibitory factor 1 (Wif-1),adenomatosis polyposis coli (Apc), dickkopf homolog 1 (Dkk1),glycogen synthase kinase 3 b (Gsk3b) and the b-catenin degrading

enzyme ubiquitin-conjugating enzyme E2B (Ube2b) were all down-regulated in Flk1þ progenitors (Table 1). Paradoxically, the Wnt/b-catenin pathway has been shown to have stage-specific effects oncardiac differentiation, both promoting and inhibiting this process[22]. Wnt/b-catenin’s divergent effects may be explained by the factthat b-catenin/CBP-mediated transcription is critical for cell prolif-eration without differentiation, whereas a switch to b-catenin/p300-mediated transcription is critical to initiate a differentiationprogram with a more limited proliferative capacity [22,26]. Toexplore these divergent b-catenin signaling pathways in Flk1þ CPCs,we utilized the small molecule antagonist IQ1 that selectivelyinhibits p300-dependent b-catenin signaling [22]. IQ1 specificallyblocks the coactivator p300 from interacting with the proteinb-catenin, a process that is required for the initiation of differenti-ation,while at the same time enhancing the interaction between the

Fig. 2. (A) D3 ES and alpha-MHC-Luc ES cells were cultured for 4 days on ColI (blue), ColIV (orange), laminin (Lam.; red) or fibronectin (Fibro.; green). (B) Quantitative real-time PCRdemonstrates a significant increase in Flk1 expression in differentiating ES cells cultured on ColIV or laminin *P < 0.05 versus undifferentiated (undiff.) ES cells; ColI- and Fibro-exposed cultures. (C) Luciferase assays show significantly higher alpha-MHC-luciferase activities in ColIV- and Lam.-exposed cultures. *P < 0.05 versus undiff. ES cells; ColI- andFibro-exposed cultures. (D) alpha-MHC-Luc ES cells were cultured for 4 days on ColIV before Flk1þ CPCs were MACS-sorted. Flk1þ CPCs were then exposed for 4 days to ColI (blue) orColIV (orange), Lam (red) or Fibro (green). (E) Luciferase assays show significantly higher alpha-MHC-dependent luciferase activity in Fibro-exposed cultures. *P < 0.05 versus alpha-MHC-Luc ES cell-derived Flk1þ CPCs; ColI-, ColIV- and Fibro.-exposed cultures. For interpretation of the references to color in this figure legend, the reader is referred to the webversion of this article.

K. Schenke-Layland et al. / Biomaterials 32 (2011) 2748e27562752

coactivator CBP and b-catenin thereby promoting the b-catenin-driven expansion of stem cells and preventing spontaneous differ-entiation [22,26]. After confirming that gene expression profiles ofpre-expansion Flk1þ CPCs were similar to Flk1þ CPCs that had beenexpanded using IQ1-supplemented medium (Suppl. Fig. 1B), wecultured ES cells on either gelatin or ColIV-coated 2D culturesurfaces in the absence or presence of IQ1 introduced at two sepa-rate developmental time points (Fig. 3B). In the first group, IQ1 wasadded at day 0 of the culture prior to commitment to the cardio-vascular lineage (pre-commitment). In group two, IQ1was added atculture day 2, after ES cells had begun to differentiate (post-commitment). When added to undifferentiated ES cells, IQ1 had aninhibitory effect on the cardiovascular differentiation potential of EScells cultured on ColIV as determined by the percentage of Flk1þ

CPCs within the cultures (ColIV (�) IQ1: 2.05 � 0.45% versus ColIV(þ) IQ1: 1.04 � 0.22%; P < 0.0001) (Fig. 3B). In contrast, whenadministered after 2 days of differentiation, post-commitment tothe cardiovascular lineage, IQ1 significantly increased thenumberofFlk1þ CPCs within the ColIV cultures (3.41 � 0.43%; P ¼ 0.016)compared to the cultures without IQ1 supplement (2.17 � 0.31%)

(Fig. 3B). These data support a model where the increase in CPCsseen with IQ1 is related to its ability to block differentiation andpromote expansion of CPCs versus promoting increased CPCsthrough enhancing ES cell differentiation to CPCs.

3.5. Recapitulation of the 3D niche microenvironment

ColIV increases the differentiation potential of mouse ES cellsinto CPCs in vitro in two-dimensional (2D) culture systems.However, ColIV is really part of a 3Dnichemicroenvironment in vivo,and thus 2D systems cannot faithfully recapitulate these morecomplex cellematrix interactions. Thus, to begin to study the effectsof three-dimensionality on CPC fate we bioengineered a 3D CPCniche in vitro employing electrospinning technologies. To fabricate3D cell culture scaffolds,10% gelatin and 10% polycaprolactone (PCL)were mixed and electrospun to create hybrid scaffolds as describedin detail previously [24]. In order to mimic not only the structuralbut also thebiological properties of the endogenousCPCniche, someof the electrospun substrates were coated with ColIV (Fig. 4A). Theultrastructure of the resulting fibrous scaffolds, both non-coated

Fig. 3. (A) Immunofluorescence staining of an E15.5 mouse heart demonstrates high levels of expression of b-catenin (green) in Isl1þ CPCs (red). DAPI-stained nuclei are shown inblue. (B) FACS of differentiating ES cells cultured on either gelatin or ColIV with or without IQ1 (4 mg/ml). *P < 0.0001 compared to ColIV pre-commitment with IQ1; **P ¼ 0.016compared to ColIV post-commitment with IQ1; ***P < 0.0001 compared to gelatin post-commitment with IQ1. (For interpretation of the references to color in this figure legend, thereader is referred to the web version of this article).

K. Schenke-Layland et al. / Biomaterials 32 (2011) 2748e2756 2753

(Fig. 4B) and ColIV-coated (Fig. 4C) are shown. The fiber size wascomparable in non-coated (1.67 � 0.45 mm) and ColIV-coatedsubstrates (1.51�0.26mm), but the averagepore sizedecreased afterColIV-coating (10.23 � 2.88 mm2 and 5.96 � 1.47 mm2). Immuno-fluorescence imaging was performed to confirm the successfulcoating of the electrospun material with ColIV (Fig. 4D and E). Toassess the impact of three-dimensionality onCPC expansion, ES cellswere cultured on either 2D coated surfaces or the 3D gelatin/PCL or3D gelatin/PCL/ColIV electrospun cell culture inserts (Fig. 4F).Similar to our previous results, 2D ColIV increased the percentage ofFlk1þ cells significantly when compared to the 2D gelatin cultures(2.57 � 0.21% compared to 0.7 � 0.17%; P ¼ 0.004). However,providing a 3D microenvironment further increased the fraction ofFlk1-expressing CPCs (3.26 � 0.37%; P < 0.0001). Introducing ColIVto the 3D scaffold, significantly enhanced this effect (7.96 � 0.43%;P < 0.0001) (Fig. 4F).

To assess if recapitulation of all aspects of the CPC niche in vitrowould have an additive effect on CPC expansion, we combined theinhibitor of p300-dependent b-catenin signaling with three-dimensionality. We exposed undifferentiated ES cells to either the2D or 3D culture systems and introduced IQ1 or vehicle to the cellculture medium after 2 days of culture. FACS analysis for Flk1expression revealed that culture on 3D ColIV-coated electrospun

Table 1Microarray analysis of differential gene expression of Wnt/b-catenin signalingpathway in ES cell-derived Flk1þ CPCs.

Gene symbol Gene name Fold increase in expressionin Flk1þ vs Flk1� cells

Wnt9a Wingless-type MMTVintegration site 9A

11.2

Wnt1 Wingless-related MMTVintegration site 1

2.3

Ctnnb1 Catenin, beta 1 1.32Sox7 SRY-box containing gene 7 3.04Fzd4 Frizzled homolog 4 2.23c-myc Myelocytomatosis oncogene 6.64Wif-1 Wnt inhibitory factor 1 0.07Apc Adenomatosis polyposis coli 0.08Dkk1 Dickkopf homolog 1 0.23Gsk3b Glycogen synthase kinase 3 beta 0.68Ube2b Ubiquitin-conjugating enzyme E2B 0.42

scaffolds and inhibition of b-catenin signaling with IQ1 wereadditive (Fig. 5). The percentage of Flk1þ CPCs in 3D ColIV-coated/IQ1-treated cultures (7.97 � 0.6%; P < 0.0001) was significantlygreater then either 3D (3.55� 0.32%) or IQ1 treatment (1.35� 0.14%(gelatin); 2.34 � 0.31% (ColIV)) alone.

4. Discussion

It has been speculated that, similar to satellite cells in adultskeletal muscle, cardiac stem cells in the adult heart are quiescentcells, surrounded by BM proteins including ColIV and laminin, andgive rise to cardiovascular cells through asymmetric division whenneeded for cardiac repair [10,17]. However, these adult cardiac stemcells differ widely from the cardiac progenitor cell populationsdescribed in the fetal heart and whether a stem or progenitor cellniche existed in the developing heart was unknown. This studyrepresents the first characterization of the CPC in vivo niche in bothfetal human and mouse hearts. We found that endogenous, multi-potent Isl1þ/Flk1þ CPCs reside within niche clusters in the rightventricular free wall, the atria and outflow tracks that were tightlycircumscribed by the BM ECM proteins ColIV and laminin. Thisassociation of Isl1þ/Flk1þ CPCswith ColIV and lamininwas also seenin differentiatingmouse ES cell-derived EBs. The ability of ColIV andlaminin to enhance CPC differentiation from mouse ES cells andsupport their expansion is consistent with our previous studies[5,20]. We also demonstrate that ECM proteins such as ColI andfibronectin surround these CPC niches within the fetal myocardiumand may selectively promote cardiovascular differentiation of CPCs.Consistent with this model, Isl1-positive CPCs that migrated awayfrom the niche, down-regulated Isl1 while committing to a CMphenotype, indicating the uniqueness of this in vivo niche micro-environment to maintain CPCs in their undifferentiated state.Interestingly, fibronectin specifically was capable of enhancing thedifferentiation of mouse ES cell-derived Flk1þ CPCs into CMs andhas previously been shown to support differentiation of vascularcells in vitro [5,25,27]. The molecular basis for the selective effect ofthese ECM proteins on CPC fate is speculative, but likely is mediatedby their interactions with CPC-expressed integrins.

Several specific factors that regulate cell fate decisions and thuscontrol normal embryonic development have been discovered and

Fig. 4. (A) Schematic of the electrospinning set-up and protocol of 2D versus 3D experiments. (BeC) Scanning electron micrographs of electrospun gelatin-PCL hybrid substrateswithout (B) or with (C) ColIV-coating. (DeE) Compared to the non-coated controls (D), immunofluorescence staining reveals the successful ColIV-coating of the electrospun material(E). (F) FACS analyses revealed an increased percentage of Flk1þ cells from 3D gelatin/PCL/ColIV cultures. *P ¼ 0.004 versus undiff ES cells and 2D gelatin cultures. **P < 0.0001versus undiff. ES cells and 2D gelatin and 2D ColIV cultures. ***P < 0.0001 versus undiff. ES cells and gelatin, 2D ColIV and 3D gelatin/PCL cultures.

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include members of the bone morphogenetic protein (BMP),hedgehog, fibroblast growth factor (FGF) and Wnt families alongwith small molecules such as retinoic acid [15,26]. Within thisgroup, Wnt proteins are particularly important as they controlnumerous functions during development such as embryonicinduction, generation of cell polarity and the specification of cellfate [26,28].Wnt signaling has also been implicated to be importantfor the maintenance and self-renewal of ES cells as well as stem/progenitor cells of various lineages, including the cardiovascularlineage [11]. Microarray analysis of CPC gene expression suggestedthat Wnt signaling through b-catenin was activated in CPCs andendogenous Isl1þ/Flk1þ CPCs within the fetal cardiovascular nicheexpressed high levels of b-catenin. Wnt/b-catenin signaling hasbeen reported to have a stage dependent, paradoxical effect oncardiac differentiation [22], but until recently, there has been noclear rationale for the dichotomous behavior of Wnt/b-cateninsignaling in promoting self-renewal and proliferation in some cellswhile enhancing differentiation in others [26]. Recent studies haveshown that b-catenin/CBP-mediated transcription is critical forstem/progenitor maintenance and proliferation without differen-tiation, whereas a switch to b-catenin/p300-mediated transcriptionis critical to initiate differentiationwith a more limited proliferativecapacity [22,26]. To define the role of b-catenin signaling in CPC

development, we utilized the small signaling molecule IQ1 thatselectively inhibits p300-dependent b-catenin signaling [22]. Wefound that IQ1 had an inhibitory effect on CPC differentiationwhenintroduced into cultures of undifferentiated mouse ES cells prior tocommitment to the cardiovascular lineage, as suggested in theoriginal reports [22]. When administered post-commitment to thecardiovascular lineage, IQ1 significantly increased Flk1þ CPCnumbers, which is likely a result of expansion and prevention ofdifferentiation of the existing pool of CPCs.

All stem and progenitor cell in vivo niches are complex 3Dmicroenvironments but the importance of this structure to itsfunction is unknown. However, awareness of the potential biolog-ical differences between 3D versus 2D environment has promptedinvestigators to bioengineer structures that mimic the 3D proper-ties of natural ECM [8,29e32]. To examine the importance of three-dimensionality on CPC fate, we created 3D CPC niche-like scaffoldswith electrospinning [24]. When cultured in a 3D microenviron-ment, the percentage of Flk1þ progenitors increased significantlycompared to the 2D cultures. By incorporating ColIV, a componentof the endogenous CPC niche in vivo, we were able to increase thenumber of CPCs further. Although this work demonstrates thatthree-dimensionality is sufficient in increasing numbers of Flk1þ

progenitors compared to conventional 2D in vitro culture systems,

Fig. 5. FACS analysis of differentiating ES cells that had been cultured on eithergelatin- or ColIV-coated cell culture surfaces (2D), or gelatin/PCL or gelatin/PCL/ColIVelectrospun scaffolds (3D), with or without the presence of IQ1 post-commitment tothe cardiovascular lineage. *P < 0.0001 compared to gelatin with IQ1; **P < 0.0001compared to ColIV with IQ1; ***P < 0.0001 compared to gelatin/PCL/ColIV withoutIQ1; ****P < 0.0001 compared to gelatin/PCL with IQ1.

K. Schenke-Layland et al. / Biomaterials 32 (2011) 2748e2756 2755

understanding of the mechanisms underlying the effect that three-dimensionality has on cell fate decisions will require furtherstudies. Enhanced cellecell and cellematrix interactions leading toimproved cell signaling in 3D cultures may play an important role[33]. Endogenous CPCs do not grow as independent units. Instead,they are surrounded in all dimensions by ECM within an isolatedniche environment that provides unique cellecell interactions,celleECM interactions, and exposure to a variety of soluble factors.These interactions within the niche have been shown to be criticalfor regulating stem cell self-renewal, proliferation, survival anddifferentiation in other tissues [34]. Cellematrix interactions aregreatly impaired on in vitro tissue culture plates, where cells areforced to adjust to an artificially flat and rigid surface; 3D matricesprovide a closer approximation to the in vivo environment. Finally,we have demonstrated that incorporating aspects of both thephysical 3D niche microenvironment and signaling pathways usingIQ1 have an additive effect on CPC expansion. This suggests thatfurther understanding of the CPC niche could facilitate additionaloptimization of synthetic niches in vitro to enhance CPC expansion,which will be a necessary prerequisite if CPCs are to be expanded tosufficient numbers to be useful in regenerative therapies.

5. Conclusion

In this study we have characterized the CPC niche in developinghuman andmouse hearts. We identified crucial niche ECM proteinsand demonstrated their biological relevance for controlling CPCfate. We further demonstrated that three-dimensionality is notonly important for CPC expansion in vitro, but that in combinationwith specific inhibition of certain b-catenin signals can enhance 3Dengineered ECM protein-based in vitro culture systems. Whilepluripotent stem cells such as ES and iPS cells can be extensivelyexpanded in culture [35], the ability to expand tissue-specific stemor progenitor cells ex vivo is extremely limited. Hence, the devel-opment of advanced in vitro culture systems that exhibit features ofnatural niche microenvironments, such as the one presented here,are critically needed both for studying the role of the niche in CPCbiology but also the advancement of the field of regenerativemedicine.

Acknowledgments

This work was supported by the NIH (5T32HL007895-10 to K.S-L.; P01-HL080111 and R01-HL094941 to W.R.M.), CIRM (RB1-01354), Laubisch and Cardiovascular Development Funds (toW.R.M.), Fraunhofer-Gesellschaft (Attract 692263 to K.S.-L.), RuthKirschstein National Research Service Award (GM007185 to B.V.H;T32HL69766 to J.M.G.) and the Alberta Heritage Foundation forMedical Research Fellowship (AHFMR to A.N.).

Appendix. Supplementary data

Supplementary data associated with this article can be found inonline version at doi:10.1016/j.biomaterials.2010.12.046.

Appendix

Figures with essential color discrimination. Figs. 2,4 and 5 in thisarticle are difficult to interpret in black and white. The full colorimages can be found in the online version, at doi:10.1016/j.biomaterials.2010.12.046.

References

[1] Kattman SJ, Huber TL, Keller GM. Multipotent flk-1þ cardiovascular progenitorcells give rise to the cardiomyocyte, endothelial, and vascular smooth musclelineages. Dev Cell 2006;11:723e32.

[2] Laugwitz KL, Moretti A, Lam J, Gruber P, Chen Y, Woodard S, et al. Postnatalisl1þ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature2005;433:647e53.

[3] Moretti A, Caron L, Nakano A, Lam JT, Bernshausen A, Chen Y, et al. Multi-potent embryonic isl1þ progenitor cells lead to cardiac, smooth muscle, andendothelial cell diversification. Cell 2006;127:1151e65.

[4] Wu SM, Fujiwara Y, Cibulsky SM, Clapham DE, Lien CL, Schultheiss TM, et al.Developmental origin of a bipotential myocardial and smooth muscle cellprecursor in the mammalian heart. Cell 2006;127:1137e50.

[5] Schenke-Layland K, Rhodes KE, Angelis E, Butylkova Y, Heydarkhan-Hagvall S, Gekas C, et al. Reprogrammed mouse fibroblasts differentiateinto cells of the cardiovascular and hematopoietic lineages. Stem Cells2008;26:1537e46.

[6] Bu L, Jiang X, Martin-Puig S, Caron L, Zhu S, Shao Y, et al. Human ISL1 heartprogenitors generate diverse multipotent cardiovascular cell lineages. Nature2009;460:113e7.

[7] Fuchs E, Tumbar T, Guasch G. Socializing with the neighbors: stem cells andtheir niche. Cell 2004;116:769e78.

[8] Votteler M, Kluger P, Walles W, Schenke-Layland K. Stem cell microenviron-ments-unveiling the secret of how stem cell fate is defined. Macromol Biosci2010;10:1302e15.

[9] Watt FM, Hogan BL. Out of Eden: stem cells and their niches. Science2000;287:1427e30.

[10] Urbanek K, Cesselli D, RotaM, Nascimbene A, De Angelis A, Hosoda T, et al. Stemcell niches in theadultmouseheart. ProcNatl AcadSciUSA2006;103:9226e31.

[11] Cohen ED, Tian Y, Morrisey EE. Wnt signaling: an essential regulator ofcardiovascular differentiation, morphogenesis and progenitor self-renewal.Development 2008;135:789e98.

[12] Qyang Y, Martin-Puig S, Chiravuri M, Chen S, Xu H, Bu L, et al. The renewal anddifferentiation of Isl1þ cardiovascular progenitors are controlled by a Wnt/beta-catenin pathway. Cell Stem Cell. 2007;1:165e79.

[13] Lin L, Cui L, Zhou W, Dufort D, Zhang X, Cai CL, et al. Beta-catenin directlyregulates Islet1 expression in cardiovascular progenitors and is required formultiple aspects of cardiogenesis. Proc Natl Acad Sci U S A 2007;104:9313e8.

[14] Cohen ED, Wang Z, Lepore JJ, Lu MM, Taketo MM, Epstein DJ, et al. Wnt/beta-catenin signaling promotes expansion of Isl-1-positive cardiac progenitor cellsthrough regulation of FGF signaling. J Clin Invest 2007;117:1794e804.

[15] Nusse R. Wnt signaling and stem cell control. Cell Res 2008;18:523e7.[16] Spinale FG. Myocardial matrix remodeling and the matrix metalloproteinases:

influence on cardiac form and function. Physiol Rev 2007;87:1285e342.[17] Leri A, Kajstura J, Anversa P, Frishman WH. Myocardial regeneration and stem

cell repair. Curr Probl Cardiol 2008;33:91e153.[18] Rodriguez-Feo JA, Sluijter JP, de Kleijn DP, Pasterkamp G. Modulation of

collagen turnover in cardiovascular disease. Curr PharmDes 2005;11:2501e14.[19] Daley WP, Peters SB, Larsen M. Extracellular matrix dynamics in development

and regenerative medicine. J Cell Sci 2008;121:255e64.[20] Schenke-Layland K, Angelis E, Rhodes KE, Heydarkhan-Hagvall S, Mikkola HK,

Maclellan WR. Collagen IV induces trophoectoderm differentiation of mouseembryonic stem cells. Stem Cells 2007;25:1529e38.

K. Schenke-Layland et al. / Biomaterials 32 (2011) 2748e27562756

[21] Hakuno D, Takahashi T, Lammerding J, Lee RT. Focal adhesion kinase signalingregulatescardiogenesisofembryonic stemcells. J BiolChem2005;280:39534e44.

[22] Miyabayashi T, Teo JL, Yamamoto M, McMillan M, Nguyen C, Kahn M. Wnt/beta-catenin/CBP signaling maintains long-term murine embryonic stem cellpluripotency. Proc Natl Acad Sci U S A 2007;104:5668e73.

[23] Schenke-Layland K, Xie J, Magnusson M, Angelis E, Li X, Wu K, et al.Lymphocytic infiltration leads to degradation of lacrimal gland extracellularmatrix structures in NOD mice exhibiting a Sjogren’s syndrome-like exo-crinopathy. Exp Eye Res 2010;90:223e37.

[24] Schenke-Layland K, Rofail F, Heydarkhan S, Gluck JM, Ingle NP, Angelis E, et al.The use of three-dimensional nanostructures to instruct cells to produceextracellular matrix for regenerative medicine strategies. Biomaterials2009;30:4665e75.

[25] Yamashita J, Itoh H, Hirashima M, Ogawa M, Nishikawa S, Yurugi T, et al.Flk1�positive cells derived from embryonic stem cells serve as vascularprogenitors. Nature 2000;408:92e6.

[26] Teo JL, Kahn M. The Wnt signaling pathway in cellular proliferation and differ-entiation: a tale of two coactivators. Adv Drug Deliv Rev 2010;62:1149e55.

[27] McCloskey KE, Stice SL, Nerem RM. In vitro derivation and expansion ofendothelial cells from embryonic stem cells. Methods Mol Biol 2006;330:287e301.

[28] Logan CY, Nusse R. The Wnt signaling pathway in development and disease.Annu Rev Cell Dev Biol 2004;20:781e810.

[29] Badylak SF, Nerem RM. Progress in tissue engineering and regenerativemedicine. Proc Natl Acad Sci U S A 2010;107:3285e6.

[30] Lund AW, Stegemann JP, Plopper GE. Inhibition of ERK promotes collagen gelcompaction and fibrillogenesis to amplify the osteogenesis of humanmesenchymal stem cells in three-dimensional collagen I culture. Stem CellsDev 2009;18:331e41.

[31] Rabbany SY, James D, Rafii S. New dimensions in vascular engineering:opportunities for cancer biology. Tissue Eng Part A 2010;16:2157e9.

[32] Tate CC, Shear DA, Tate MC, Archer DR, Stein DG, LaPlaca MC. Laminin andfibronectin scaffolds enhance neural stem cell transplantation into the injuredbrain. J Tissue Eng Regen Med 2009;3:208e17.

[33] Vorotnikova E, McIntosh D, Dewilde A, Zhang J, Reing JE, Zhang L, et al.Extracellular matrix-derived products modulate endothelial and progenitorcell migration and proliferation in vitro and stimulate regenerative healing invivo. Matrix Biol 2010;29:690e700.

[34] Krause DS. Regulation of hematopoietic stem cell fate. Oncogene 2002;21:3262e9.

[35] Hochedlinger K, Plath K. Epigenetic reprogramming and induced pluri-potency. Development 2009;136:509e23.